DESCRIPTION: The investigator has recently cloned and sequenced a protein, known as KCC1, that catalyzes the cotransport of potassium and chloride. Functional studies had indicated the presence of such a protein in red cells and a few other tissues, but the investigator has produced the first molecular-level information about this transporter. The specific aims of the project are 1) determine regions of the protein important in ion binding and loop-diuretic inhibition using site-directed mutagenesis and chimeras between the K-Cl cotransporter and the Na-K-2Cl cotransporter; 2) characterize regulation of KCC-1 by determining sites of phosphorylation, detecting proteins that interact with KCC1, and characterizing the regulatory role of intracellular chloride; 3) localize KCC1 in mammalian tissues, with focus on absorptive epithelia; and 3) produce large amounts of a related cyanobacterial cation-chloride cotransporter, CCCcy, for the purpose of producing crystals suitable for structural analysis.